Lipoproteins have from since been fractionated by ultracentrifugation operation into high density lipoprotein (HDL, density: 1.063-1.21), low density lipoprotein (LDL, density: 1.019-1.063), very low density lipoprotein (VLDL, density: 1.006-1.019), and chylomicron (CM, density&lt;1.006). The fractionation operation needs an ultracentrifuge and in addition must continue centrifugation for several days so that it is impossible to treat many samples.
In place of this, a method has become the main current in which by mixing serum with a solution which comprises a precipitation agent that contains both a polyethylene glycol or a polyanion such as dextransulfate and a divalent cation such as calcium, or both phosphorous tungstic acid and a divalent cation, LDL, VLDL, and CM are precipitated and only HDL that remains in the supernatant after the centrifugation is fractionated. In this method, use can be made of an automatic analyzing apparatus which has widely prevailed in the field of clinical assays. That is, since the assay of total cholesterol using an enzyme assay method has been established by use of an automatic analyzing apparatus, utilization of the established assay method has enabled the measurement of concentration of cholesterol in the fractionated HDL.
However, this method still needs centrifugation operation though at a low speed and there has been the problem of artificial errors in quantitative determination or misplacement of samples when the precipitation agent and serum are mixed with each other. In addition, it has been impossible to measure other general biochemical items simultaneously by use of an automatic analyzing apparatus. Clinical assays are demanded to be carried out speedily, which also give rise to the desire for a shortening of test time.
On the other hand, from clinical point of view, there has been a report which lays importance on the level of cholesterol in LDL, which is a risk factor for arteriosclerosis ("Standard Level of Total Cholesterol and Basis for Its Setting", Domyaku Koka (Arteriosclerosis), 24 (6), 260 (1966)). Currently, the level of cholesterol in LDL is obtained from measured values of total cholesterol (T-CHO), neutral fats (TG) and cholesterol in HDL according to an equation by introducing an empirical factor therein. The equation (Friedewald W. T., et al., Clin. Chem., 18, 499-502 (1972)) is as shown below; EQU Cholesterol value in LDL=(T-CHO value)-(Cholesterol value in HDL)-(TG value)/5.
In this method, all the three terms to be measured must be exactly measured before the equation can be valid. It is said that the calculated value does not reflect the concentration of cholesterol in LDL when TG value exceeds 400 mg/dl or the concentration of cholesterol in LDL is 100 mg/dl or less (Warnick G. R., et al., Clin. Chem., 36(1), 15-19 (1990), McNamara, J. R., et al., Clin. Chem., 36(1), 36-42 (1990)) Therefore, extraordinary values which are the target of assay cannot be attained by this method. Besides, there have been a method in which lipoprotein is isolated by electrophoresis and the quantity of protein is measured or a method in which cholesterol is measured for respective lipoproteins by HPLC. However, both of them are deficient in the ability to treat many samples and need an expensive apparatus.
Recently, in order to dissolve the above-described problems associated with the measurement of cholesterol in HDL, an automatic kit for the measurement of cholesterol in HDL has been developed and prevailing. However, the technologies described in the Japanese Registered Patent No. 2600065, Japanese Patent Application Laid-open No. Hei 8-201393 and Japanese Patent Application Laid-open No. Hei 8-31195 use a precipitation agent in combination and the metal used as a divalent cation contained in the precipitation agent forms insoluble precipitation by the action of a detergent generally used in an automatic analyzing apparatus, and the precipitation accumulates in a waste disposal mechanism, which causes a disorder of the automatic analyzing apparatus. Further, insoluble agglutinates are formed in the reaction mixture and the agglutinates not only make the reaction mixture turbid, which could affect the results of measurement, and cause an error in measurement, but also stain the reaction cell to give not a small influence on the results of measurement of other biochemical items simultaneously measured.
In most automatic analyzing apparatus now prevailing, the reaction is often completed in 10 minutes. In this case, the occurrence of a change in turbidity, if any, casts a question on the accuracy of the measured values. In addition, the problem also arises that the turbidity of reaction mixture deteriorates reproducibility. Therefore, samples which can be measured in the apparatus are limited and a wide range of measuring wavelengths and various types of samples from patients cannot be served. For example, it has the defect that in the vicinity of 340 nm (UV region), the absorbance is 2 or more, or 3 or more and frequently exceeds the tolerance limits of the analyzing apparatus, due to the turbidity caused by the agglutinates.
The technology described in Japanese Patent Application Laid-open No. Hei 9-96637, which uses a divalent cation, is a method involving use of an antiserum contained in the reaction mixture which forms agglutinates with a lipoprotein. This also forms insoluble antigen-antibody agglutinates so that the reaction cell is stained. Therefore, no small an influence is given on the results of measurement of other biochemical items that are measured simultaneously. Furthermore, the high turbidity in the reaction mixture makes accurate measurement impossible for the cholesterol in HDL in particular in UV region because of the same causes as described above. In a longer wavelength region, the measured values are inaccurate due to the influence of turbidity.
On the other hand, in the case of measurement of cholesterol in LDL, it is a current status that a calculation method has to be taken.
An object of the present invention is to provide a method for assaying biological specimens for substances contained in the components of each specimen, such as cholesterol in HDL or LDL, etc., by use of a general-purpose automatic analyzing apparatus, without separation of the specimen by centrifugation operation and to provide a reagent for use therein.